Evidence for the participation of Cys sub 558 and Cys sub 559 at the active site of mercuric reductase

Journal Article · Tue Feb 07 04:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00429a037· OSTI ID:5471827

Miller, S M; Moore, M J; Massey, V; Williams, Jr, C H; Distefano, M D; Ballou, D P; Walsh, C T ^[1]

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1. Univ. of Michigan, Ann Arbor (USA)

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH{sub 2} form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH{sub 2} has previously been reported to require only 2 electrons for reduction of the active site disulfide. The authors present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH{sub 2} actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4{degree}C and at 25{degree}C indicate that reduction of the active site occurs rapidly, as previously reported; this is followed by a slower reduction of another redox group via reaction with the active site. ({sup 14}C)Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys{sub 558} and Cys{sub 559}, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, E{sub ox} with Cys{sub 558} and Cys{sub 559} as thiols exhibits less than 50% of the fluorescence of E{sub ox} where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site. Initial velocity measurements show that the auxiliary disulfide must be reduced before catalytic Hg(II) reduction can occur, consistent with the report of a preactivation phenomenon with NADPH or cysteine. A modified minimal catalytic mechanism is proposed as well as several chemical mechanisms for the Hg(II) reduction step.

• OSTI ID: 5471827
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:3; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
560300 -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
COENZYMES
CYSTEINE
ENZYMES
ESCHERICHIA COLI
HALIDES
HALOGEN COMPOUNDS
KINETICS
LABELLED COMPOUNDS
MERCURY CHLORIDES
MERCURY COMPOUNDS
MERCURY HALIDES
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
REACTION KINETICS
REDOX REACTIONS
REDUCTION
TEMPERATURE EFFECTS
THIOLS